Interferons as modulators of human monocyte-macrophage differentiation. I. Interferon-gamma increases HLA-DR expression and inhibits phagocytosis of zymosan

The development of HLA-DR (Ia) expression in the presence and absence of interferon-gamma was monitored in monocyte-macrophage cultures. Overnight incubation with doses as low as 5 U/ml gave elevated values for Ia expression and the maximum increase was obtained with 200 U/ml. In contrast interferon-alpha had only a slight effect on the expression of Ia at doses as high as 2000 U/ml. The increase seen at 24 hr was maintained during the first 2 days of culture. The interferon-gamma-treated cells expressed four to five times more Ia than fresh monocytes. During the same time, monocytes cultured in the absence of interferon expressed approximately two times the amount of fresh monocytes. When the surface density of Ia was calculated, the interferon-gamma-treated monocytes expressed twice that of the untreated cells. Major changes in morphology and size occurred between days 3 and 4 of monocyte to macrophage development. Consequently a rapid increase in Ia expression took place; however, when the surface density was calculated this value increased only slightly when the monocytes matured to macrophages. The interferon-gamma-treated cells continued to express more total Ia as well as having increased surface density of this antigen. Interferon-gamma was also added to monocyte-macrophages several days after culture initiation (days 3, 4, and 5). Despite being in different stages of maturation, the cells responded to the interferon with increased Ia expression and surface density. The phagocytic activity of opsonized zymosans was also monitored. In contrast to Ia expression, this activity was downregulated by interferon-gamma, and the lower levels of phagocytosis were maintained through the 7 days of observation. Thus, interferon-gamma appears to change the differentiation pathway of the monocyte. The signal stimulates an increased level of Ia that may assist in the initiation of immune responses, and at the same time downregulates the scavenger role of removing opsonized particles. Once the monocyte has received this specific signal it continues to develop in a pathway different from that of the nontreated monocytes.     

Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free cultures

The role of mononuclear phagocyte-specific colony-stimulating factor (CSF-1) in human monocyte to macrophage differentiation was investigated. The addition of 1000 U/ml of CSF-1 to serum-free monocyte cultures resulted in monocyte survival comparable to that in cultures containing 5% AB serum, whereas cells in serum- and CSF-1-free medium lost their viability in 3 to 5 days. The requirement for CSF-1 coincided with the time (40 to 64 hr of culture) when the major changes in morphology and biochemical function took place in monocytes undergoing differentiation into macrophages. If CSF-1 was removed from the cultures before this time, death of the monocytes resulted. In cultures containing CSF-1, as in serum containing cultures, the lysosomal enzyme acid phosphatase was enhanced 10- to 20-fold by day 4 to 5. Superoxide production in response to phorbol myristic acetate was maintained in CSF-1 cultured monocytes, but declined with time in monocytes cultured in serum. The expression of monocyte-macrophage antigens p150.95 (LeuM5), OKM1, LeuM3, Fc receptors (32.2), and HLA-DR had increased in CSF-1 containing cultures at day 4. When antigen expression was analyzed at day 2 to 3, when cell size and 90 degrees scatter characteristics were still identical to control serum-free cultures, only p150.95, HLA-DR and FcR expression were enhanced by CSF-1. Low amounts of lipopolysaccharide (0.1 ng/ml) were found to enhance monocyte survival in the absence of added CSF-1. Lipopolysaccharide-containing cultures were found to produce CSF-1 (up to 450 U/ml, as detected by radioimmunoassay). Lipopolysaccharide (1 microgram/ml), however, did not induce enhanced expression of the maturation-related antigens. Based on these observations we conclude that CSF-1 is enhancing human monocyte survival and is involved in the events leading to the differentiation of monocytes into macrophages.     

Examination of survival, proliferation and cell surface antigen expression of human monocytes exposed to macrophage colony-stimulating factor (M-CSF)

The effects of macrophage colony-stimulating factor (M-CSF or CSF-1) on the survival, proliferation, maturation and activation of human blood monocytes were examined. M-CSF (100-1,000 U/ml) doubled the number of monocytes surviving after eight days in culture and accelerated the usual increase in cell volume. Antiserum to M-CSF abolished both of these effects. There was no sizable increase in 3H-thymidine incorporation in monocytes over this time period. Of various factors tested, including gamma-interferon (gamma-IFN), interleukin (IL) 1 alpha, granulocyte CSF (G-CSF), platelet-derived growth factor (PDGF), and lipopolysaccharide (LPS), only granulocyte-macrophage CSF (GM-CSF) could also enhance survival and augment cell volume. While antiserum to human M-CSF eliminated the increase in survival induced by GM-CSF, it could not ablate the GM-CSF-stimulated increase in monocyte cell volume. Monocyte cell surface markers that increase with maturation (i.e., Fc gamma RIII) or with activation (i.e., Fc gamma RI) were unaffected by incubation with M-CSF.     

Population kinetic study on the origin of guinea pig monocyte heterogeneity

Population kinetic studies were performed on guinea pig peripheral blood monocyte fractions isolated by counter-flow centrifugation elutriation following a single in vivo pulse of tritiated thymidine. Labeled large monocytes (volume 317 micron3; relative distribution 49%; circulating half-life 5.7 hr; and production rate 17,000 cells/ml blood/hr) accumulated in the circulation more rapidly, had a faster turnover time, and were produced in greater numbers than small monocytes (volume 283 micron3; relative distribution 34%; circulating half-life 10.8 hr; and production rate of 6100 cells/ml blood/hr). The kinetic data do not support a maturational sequence of small into large monocytes. Intermediate monocytes (volume 300 micron3; relative distribution 11%; circulating half-life 18.2 hr) and very large monocytes (volume 354 micron3; relative distribution 6%; circulating half-life 36.5 hr) had production rates, respectively, of only 1200 and 320 cells/ml blood/hr. Maxima in the labeling index curve for small and large monocytes suggested a generation time of 24 hr while grain count analysis revealed that these two cell fractions were derived from a precursor population with similar numbers of reductive divisions. Grain count analysis of intermediate and very large monocytes revealed that these cells differed from both small and large monocytes. Our data support the concept that monocyte subsets exist in guinea pig peripheral blood with different kinetics of production and survival.     

Improving the methods for isolation of monocyte and establishing macrophage cell culture in caprine model

Monocytes are widely used for immunological research, especially in the study of innate immune system. Although methods for isolation of human monocytes have been established, the procedure for non-human monocyte has not been well developed. This paper describes an improved method for isolation of monocyte and the subsequent macrophage cultivation from caprine blood. Monocytes were isolated from 16 ml of heparinized caprine blood using double density methods; the Ficoll and Percoll. The number of monocytes obtained was 5.12 ± 0.89 × 10⁷ cells/ml at 70 % purity. The isolated monocytes were maintained in 10 % fetal bovine serum-enriched Dulbecco’s Modified Eagle Medium for maturation to form macrophage cell culture. At the end of the experiment, the harvested macrophage was 2.48 ± 0.33 × 10⁶ cells/ml.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Hydrocortisone-mediated inhibition of monocyte antigen presentation: Dissociation of inhibitory effect and expression of DR antigens

The suppressive effects of hydrocortisone (HC) on the human immune system are well known. The mediation of the immunosuppressive effects of HC on lymphocyte responses via inhibition of monocyte function has been examined by monocyte-dependent, antigen-induced lymphocyte proliferation. Monocytes that were first treated with HC and then washed were unaffected in their subsequent ability to present antigen. However, there was a dramatic inhibition of lymphocyte proliferative responses if HC was present while monocytes were pulsed with antigen. This was directly related to the dose of HC present. HC-mediated inhibition of monocyte antigen presentation could not be overcome by the addition of interleukin-1 (IL-1) to the cultures, and thus inhibition of monocyte IL-1 secretion cannot totally account for the inhibition of monocyte antigen presentation. Although HC inhibits monocyte antigen presentation, HC increases the expression of HLA-DR antigens on monocytes. Other monocyte stimulants, including lipopolysaccharide (LPS), lymphokine, and gamma interferon, were examined for their effect on monocyte DR expression and their effect on monocyte antigen presentation. No correlation was found between the ability to increase monocyte DR antigen expression and the effect on antigen presentation. While HC, lymphokine, and gamma interferon all increased the expression of DR antigens on monocytes, HC, LPS, and lymphokine, but not gamma interferon, inhibited monocyte antigen presentation. Although HC can exert profound immunosuppressive effects via monocytes, it is not the only mechanism of inhibition. HC added to cultures after monocytes had been pulsed with antigen was also inhibitory.     

Interferon-gamma enhances target cell sensitivity to monocyte killing

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.     

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.     

Lymphokine-mediated activation of human monocytes: neutralization by monoclonal antibody to interferon-gamma

Purified natural and recombinant human immune interferon (IFN-gamma) were found to activate human monocytes from peripheral blood to exert enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells. A marked monocyte activation was observed at low concentrations (1 and 10 U/ml) of IFN-gamma. Marked monocyte activation was also obtained with two lymphokine preparations, produced in peripheral blood mononuclear cell (PBM) cultures induced with phytohemagglutinin (PHA) or by combined stimulation with PHA and 12-O-tetradecanoylphorbol 13-acetate (TPA). The component responsible for macrophage activation in such lymphokine preparations in the past was considered to be "macrophage-activating factor" (MAF). When monoclonal antibody specifically neutralizing IFN-gamma was added to these lymphokine preparations, all MAF activity disappeared, indicating that IFN-gamma is the sole protein showing MAF activity in these preparations.     

Induction of differentiation of the human histiocytic lymphoma cell line U937 in the absence of vimentin expression

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.     

Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.     

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.     

Infection of monocytes/macrophages by HIV in vitro

Because of the very important role of the mononuclear phagocyte system in the immunopathogenesis of HIV infection, a culture system for in vitro studies of infection of monocytes/macrophages with HIV was developed. A method is described for the infection of human monocytes/macrophages cultured on hydrophobic membranes (Teflon) with different strains of HIV. The HIV isolates can be characterized according to their replication potential on monocyte/macrophages cultures. The biological properties of some HIV1 and HIV2 isolates are compared in lymphocyte and monocyte/macrophage cultures.     

Expression of functional IL 2 receptors by lipopolysaccharide and interferon-gamma stimulated human monocytes

Human peripheral blood monocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) alone or in combination. Stimulated but not resting monocytes displayed the Tac peptide of the interleukin 2 (IL 2) receptor within 24 hr as measured by immunofluorescence staining and [3H] Tac binding. The total number of anti-Tac binding sites on co-stimulated monocytes was 13,700. By using scatchard analysis with radiolabeled IL 2, the activated cells were shown to express low numbers (below 100 sites/cell) of high affinity binding sites with a KD of approximately 15 pM. LPS and IFN-gamma were additive in augmenting the number of IL 2 and anti-Tac binding sites. By using an ELISA assay specific for the soluble released form of the Tac peptide we identified 112 U/ml of IL 2 receptors in the supernatant of monocytes stimulated for 24 hr with IFN-gamma, 233 U/ml after stimulation with LPS, and 519 U/ml after the addition of both stimulating agents. Both the membrane form (55,000 daltons), as well as the soluble form (45,000 to 50,000 daltons) of the Tac, IL 2 receptor, peptide from monocytes were shown by immunoprecipitation and gel electrophoresis to be similar size to the comparable forms of these receptors derived from activated T cells. In addition, monocytes stimulated for 8 hr contained mRNA specifically hybridizing to a cDNA probe coding for the Tac peptide. Finally, activated monocytes responded to the addition of recombinant IL 2 by an increase in H2O2 production that was measured by using fluorescent indicator 2,7-dichlorofluorescein. This response as well as the observed induction of monocytic IL 2 receptors by LPS may point to a functional role for this receptor during monocyte/macrophage responses to microbial infections.     

Human mononuclear phagocyte-associated antigens: I. Identification and characterization

Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMØ). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMØ. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMØ with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.